Method for purifying an interferon

ABSTRACT

A method of purifying a polypeptide having a physiological activity such as one having interferon activities from a culture mixture of a microorganism obtained by a recombinant DNA technique and capable of producing the polypeptide is disclosed. The method comprises subjecting the cultured cells to extraction and purification in the presence of a salt of zinc or copper and polyethyleneimine thereby inhibiting decomposition and denaturation of the polypeptide. The extracted polypeptide can be further purified by column chromatographies using a column containing an anion exchange resin, column containing a cation exchange resin and column containing a gel filtration resin.

TECHNICAL FIELD

This invention relates to a method for purifying a physiologicallyactive polypeptide produced by a recombinant DNA technology withoutdenaturation or decomposition by proteases, more particularly, saidpolypeptide being produced by a microorganism transformed by a plasmidvector bearing a gene coding for a polypeptide having physiologicalactivities. This Invention in particular provides an effective methodfor purifying a desired polypeptide from a culture mixture of amicroorganism capable of producing a polypeptide having interferonactivities, especially human immune (or gamma) interferon activities.

BACKGROUND ART

Interferon proteins have been classified into three types, alpha, betaand gamma (abbreviated to IFN-α, IFN-β and IFN-γ respectively) based onantigenic and structural differences. Gamma interferon has a number ofcharacteristics that differentiate it from alpha and beta interferons.Among these differences are antigenic distinctiveness and greateractivity with regard to immunoregulation and anti-tumor effects. Humangamma interferon (referred to herein as "h-IFN-γ") may be produced by Tlymphocytes stimulated by mutagens or by antigens to which they aresensitized. It may also be obtained through cloning and expressiontechniques now well known to the art.

Recently, it has become possible by the progress in genetic engineeringto produce many physiologically active polypeptides from microorganismsor animal cells, although these substances have been produced byseparation and purification from an organism. However, it cannot yet besaid that a method has been established for extracting and purifying theintended substance with a purity sufficient to be used for drugs andwithout causing denaturation or decomposition.

Gamma interferon-containing cells, however obtained, are collected andare disrupted by various means such as osmotic shock, ultrasonicvibration, grinding or high shear disruption and the disruptedcell-gamma interferon mixture is then processed to isolate the gammainterferon. The insoluble debris is separated by centrifugation and thegamma interferon-containing supernatant is collected for purification.

Although disclosure has been made of certain technology for suchproduction methods, e.g. a method extracting and purifying thepolypeptide produced by recombinant microorganism by using guanidinehydrochloride and urea (Japanese Patent Public Disclosure No.161321/1984 and U.S. Pat. No. 4,476,049) and a purification method usinga monoclonal antibody (Japanese Patent Public Disclosure No.186995/1984), the intended substance is not always adequately purifiedwithout being subjected to denaturation and without its activity beinglost.

European Patent Application 0,087,686 discloses a three-step process forpurifying human immune interferions from the cell-free supernatant orextract from the crude interferon source. In the first step (fornaturally occurring interferon), an affinity column, such asConcanavalin-A Sepharose is used, followed by chromatography on acarboxymethyl silica column using an increasing salt gradient andfinally, on a silica gel permeation column. If sufficient purity is notobtained, concentration and chromatography on either the TSK or CMcolumn is used.

European Patent Application 0,063,482 disclosed a purification processemploying chromatographic methods using (1) Controlled Pore Glass beads;(2) Concanavalin-A Sepharose; (3) Heparin-Sepharose or ProcionRed-agarose; and (4) gel filtration.

European Patent Applications 0,107,498 and 0,077,670 disclose apurification scheme employing (1) polyethyleneimine precipitation; (2)pH precipitation of bacterial proteins; (3) concentration and dialysis;(4) chromatography on (a) carboxymethyl cellulose; (b) a calciumphosphate gel; (c) a carboxymethyl cellulose; and (d) gel filtrationresins.

These purification processes require a multitude of steps, causedegradation of the interferon by degradation or aggregation of theinterferon molecule, or otherwise result in a gamma interferon productobtained in low yield or with low activity.

It goes without saying that a method for extracting and purifying theintended polypeptide from the culture mixture of the intendedsubstance-producing microorganism without the activity of the intendedsubstance being lost and without being accompanied by denaturation isimportant for such used as pharmaceuticals and that the establishment ofsuch technology is useful from the viewpoint of industry.

Such a purification method has been particularly desired for interferon,the employment of which in pharmaceuticals is now proceeding.Interferons have anti-virus activity, but IFN-γ is expected to be usefulas an anti-tumor agent and immune regulator because of its particularlystrong cell growth inhibition. Furthermore, interferon activity hasseveral specificities; for example, when interferon is used as apharmaceutical, it is preferable to use interferon which originated froma human. Furthermore, it is desirable to establish processes forextracting and purifying interferon produced by genetic engineering.

Usually, in extracting and purifying a polypeptide obtained fromrecombinant microorganisms, cultured microorganisms are first killed byusing a bactericide (a necessary process from the viewpoint of safety)and then the dead cells are disrupted and subsequently subjected toextraction. In these treatments, the intended polypeptide is sometimesdenaturated and its activity may be lost. Furthermore, these treatmentsare apt to activate proteases included in the cells and sometimesdecompose the intended polypeptide.

A method in which a protein denatured and solubilized with a denaturingagent such as urea or guanidine hydrochloride is extracted and thedenaturing agent is removed in the course of purification has previouslybeen disclosed for polypeptides purified from cells or recombinantmicroorganisms (as described before in Japanese Patent Public DisclosureNo. 161321/1984, U.S. Pat. No. 4,476,049, etc.). However, it isdifficult to safely obtain complete renaturation of the intendedpolypeptide even though the denaturing agents are removed. Therefore,this method is not preferred if the intended polypeptide is used as apharmaceutical because, when the partially denatured polypeptide ismixed, it can become an antigen. On the other hand, in the purificationmethod using a monoclonal antibody which has also been reported (asdescribed before in Japanese Patent Public Disclosure No. 186995/1984,etc.), it can be thought that a denatured and undesirable polypeptide ora polymerized polypeptide such as dimer and trimer may be bonded to themonoclonal antibody, depending upon the antigenic determinant recognizedwith the monoclonal antibody used. Recently, a method for extractingh-IFN-γ produced by recombinant Escherichia coli in the presence of aprotease inhibitor for the purpose of inhibiting decomposition ofpolypeptide with protease has been disclosed in the above mentioned U.S.Patent, but the guanidine hydrochloride used therein is also known as aprotein denaturing agent (see, example, Japanese Patent PublicDisclosure No. 161321/1984). It is therefore expected that, although thedecomposition of polypeptide with protease can be inhibited, productionof a denatured protein may well result.

It would be further desirable to (1) provide a purification scheme toseparate gamma interferon from the cell debris of the disrupted cells inwhich the gamma interferon was produced; (2) separate gamma interferonfrom cell contaminants in high yields and with high purity and activity;(3) separate recombinant gamma interferon from cell contaminants; and(4) separate gamma interferon from cell contaminants withoutsubstantially degrading the interferon. The purification processdescribed below is such a process.

DISCLOSURE OF INVENTION

The purification method of the present invention efficiently provides apolypeptide having the intended physiological activity in substantiallypure form, inhibiting decomposition of the polypeptide with protease andavoiding the denaturation of the polypeptide. Furthermore, although thepresent invention will achieve the purification of a polypeptide havingh-IFN-γ activity, the method of the present invention is also applicableto the extraction and purification of polypeptides other than h-IFN-γ,which have a site susceptible to protease decomposition, such as Arg-Lysand Arg-Arg, and are produced by recombinant microorganisms.

In the present invention, the above-described problems are solved byadding one or more salts of zinc or copper and polyethyleneimine(abbreviated to PEI below) in the extraction process. More specifically,the invention comprises suspending the culture cells of a recombinantmicroorganisms in a buffer solution containing one or more salts of zincor copper, disrupting the cells, then adding PEI to the centrifugedsupernatant, and subsequently subjecting it to a suitable purificationprocess.

Various compounds which can be used as the salts of zinc or copperinclude zinc chloride, zinc sulfate, zinc acetate, zinc acetylacetonate,and copper sulfate, but zinc chloride, zinc acetate and copper sulfateare preferred. There are differences in the optimum concentration ofsalt depending on the peptide-producing strain, but it is generally inthe range of 0.5-5 mM, more preferably 1-3 mM in the case of zinc salts,and 0.01-3 mM, more preferably 0.25-1 mM in the case of copper salt.

These salts are mixed with a buffer solution in the above-describedconcentration, the culture cells are suspended in the resulting solutionand then disrupted, and the supernatant is obtained by centrifugation.PEI is added to the supernatant to achieve a final concentration of0.5-1.1%. The addition of PEI also functions to precipitate aconsiderable amount of impure proteins. After PEI addition, thesupernatant is allowed to stand at a low temperature, for example about4° C. After centrifugation to remove the precipitates, the desiredsubstance is purified by a conventional method. The purification can beconveniently carried out by combining several columns and dialyses. Insome cases, salting out may be involved in th process. Specificembodiments will be explained below in the Examples.

Prior to the present application, there was a report about the additionof a zinc salt to the culture medium in IFN-β production with a view toincreasing productivity (Japanese Patent Public Disclosure No.146597/1984). That report was however intended to increase production intiter during the cultivation and did not describe the addition of thesalt together with PEI in the extracting step, as in the presentinvention. With regard to the use of copper compounds in thepurification process, an example of the use of a copper-chelate resincolumn was reported in Japanese Patent Public Disclosure No.167597/1984. However, that invention related to a purification methodfor a preliminarily purified IFN solution. Previous reports such asthese above inventions are essentially different from the presentinvention which is characterized by the addition of the salts in theextraction stage for the purpose of purification without causingdenaturation or decomposition of a protein throughout. Another object ofthe present invention is to provide a substantially pure polypeptidehaving h-IFN-γ activity which can be obtained by the method ofpurification and extraction of the present invention.

The construction of W3110/pIN5T4 which is one of the stains capable ofproducing a polypeptide having h-IFN-γ activity is disclosed in EuropeanPatent Application 0,134,673. The polypeptide produced by that strain iscalled GIF146 and is represented by the following amino acid sequence(I).

    ______________________________________                                        1                                                10                           Cys  Tyr    Cys    Gln  Asp  Pro  Tyr  Val  Lys  Glu                                                                           20                           Ala  Glu    Asn    Leu  Lys  Lys  Tyr  Phe  Asn  Ala                                                                           30                           Gly  His    Ser    Asp  Val  Ala  Asp  Asn  Gly  Thr                                                                           40                           Leu  Phe    Leu    Gly  Ile  Leu  Lys  Asn  Trp  Lys                                                                           50                           Glu  Glu    Ser    Asp  Arg  Lys  Ile  Met  Gln  Ser                                                                           60                           Gln  Ile    Val    Ser  Phe  Tyr  Phe  Lys  Lue  Phe                                                                           70                           Lys  Asn    Phe    Lys  Asp  Asp  Gln  Ser  Ile  Gln                                                                           80                           Lys  Ser    Val    Glu  Thr  Ile  Lys  Glu  Asp  Met                                                                           90                           Asn  Val    Lys    Phe  Phe  Asn  Ser  Asn  Lys  Lys                                                                           100                          Lys  Arg    Asp    Asp  Phe  Glu  Lys  Leu  Thr  Asn                                                                           110                          Tyr  Ser    Val    Thr  Asp  Leu  Asn  Val  Gln  Arg                                                                           120                          Lys  Als    Ile    His  Glu  Leu  Ile  Gln  Val  Met                                                                           130                          Ala  Glu    Leu    Ser  Pro  Ala  Ala  Lys  Thr  Gly                                                                           140                          Lys  Arg    Lys    Arg  Ser  Gln  Met  Leu  Phe  Arg                                                       146                                              Gly  Arg    Arg    Ala  Ser  Gln                 (I)                          ______________________________________                                    

The GIF146-producing strain is Escherichia coli W3110 transformed by aplasmid vector bearing a DNA fragment coding for the above-describedGIF146 and represented by the DNA sequence (II).

    __________________________________________________________________________    TGC TAC TGC CAG GAC CCA TAC GTG AAG GAA                                       ACG ATG ACG GTC CTG GGT ATG CAC TTC CTT                                       GCT GAA AAC CTG AAG AAA TAC TTC AAC GCT                                       CGA CTT TTG GAC TTC TTT ATG AAG TTG CGA                                       GGT CAT TCT GAC GTT GCT GAC AAC GGT ACT                                       CCA GTA AGA CTG CAA CGA CTG TTG CCA TGA                                       CTG TTC CTG GGT ATC CTG AAA AAC TGG AAA                                       GAC AAG GAC CCA TAG GAC TTT TTG ACC TTT                                       GAA GAA TCT GAC CGT AAA ATC ATG CAG TCT                                       CTT CTT AGA CTG GCA TTT TAG TAC GTC AGA                                       CAG ATC GTT TCT TTC TAC TTC AAG CTG TTC                                       GTC TAG CAA AGA AAG ATG AAG TTC GAC AAG                                       AAA AAC TTC AAG GAC GAC CAG TCT ATC CAG                                       TTT TTG AAG TTC CTG CTG GTC AGA TAG GTC                                       AAA TCT GTT GAA ACT ATC AAG GAA GAC ATG                                       TTT AGA CAA CTT TGA TAG TTC CTT CTG TAC                                       AAC GTT AAG TTC TTC AAC TCT AAC AAG AAA                                       TTG CAA TTC AAG AAG TTG AGA TTG TTC TTT                                       AAG CGT GAC GAC TTC GAA AAG CTT ACT AAC                                       TTC GCA CTG CTG AAG CTT TTC GAA TGA TTG                                       TAC TCT GTT ACT GAC CTT AAT GTA CAG CGT                                       ATG AGA CAA TGA CTG GAA TTA CAT GTC GCA                                       AAA GCT ATC CAT GAA CTG ATC CAG GTT ATG                                       TTT CGA TAG GTA CTT GAC TAG GTC CAA TAC                                       GCT GAA CTG TCC CCG GCT GCT AAA ACT GGT                                       CGA CTT GAC AGG GGC CGA CGA TTT TGA CCA                                       AAG CGT AAA AGA TCT CAG ATG CTG TTC CGT                                       TTC GCA TTT TCT AGA GTC TAC GAC AAC GCA                                       GGT CGT CGT GCT TCT CAG TAA                                                   CCA GCA GCA CGA AGA GTC ATT         (II)                                      __________________________________________________________________________

On the other hand, the strain (W3110/pIN5T4N143) capable of producing apolypeptide having h-IFN-γ activity and represented by the followingamino acid sequence (III) can be produced as explained below withreference to Examples. This polypeptide is referred to as GIF143hereunder.

    ______________________________________                                        Gln  Asp    Pro    Tyr  Val  Lys  Glu  Ala  Glu  Asn                          Leu  Lys    Lys    Tyr  Phe  Asn  Ala  Gly  His  Ser                          Asp  Val    Ala    Asp  Asn  Gly  Thr  Leu  Phe  Leu                          Gly  Ile    Leu    Lys  Asn  Trp  Lys  Glu  Glu  Ser                          Asp  Arg    Lys    Ile  Met  Gln  Ser  Gln  Ile  Val                          Ser  Phe    Tyr    Phe  Lys  Leu  Phe  Lys  Asn  Phe                          Lys  Asp    Asp    Gln  Ser  Ile  Gln  Lys  Ser  Val                          Glu  Thr    Ile    Lys  Glu  Asp  Met  Asn  Val  Lys                          Phe  Phe    Asn    Ser  Asn  Lys  Lys  Lys  Arg  Asp                          Asp  Phe    Glu    Lys  Leu  Thr  Asn  Tyr  Ser  Val                          Thr  Asp    Leu    Asn  Val  Gln  Arg  Lys  Ala  Ile                          His  Glu    Leu    Ile  Gln  Val  Met  Ala  Glu  Leu                          Ser  Pro    Ala    Ala  Lys  Thr  Gly  Lys  Arg  Lys                          Arg  Ser    Gln    Met  Leu  Phe  Arg  Gly  Arg  Arg                          Ala  Ser    Gln                                  (III)                        ______________________________________                                    

In this amino acid sequence, Gln* represents Gln or p-Gln.

Furthermore, the DNA fragment shown by the following DNA sequence codingfor the polypeptide (GIF143) may be used for production of the intendedplasmid vector.

    __________________________________________________________________________    CAG GAC CCA TAC GTG AAG GAA GCT GAA AAC                                       GTC CTG GGT ATG CAC TTC CTT CGA CTT TTG                                       CTG AAG AAA TAC TTC AAC GCT GGT CAT TCT                                       GAC TTC TTT ATG AAG TTG CGA CCA GTA AGA                                       GAC GTT GCT GAC AAC GGT ACT CTG TTC CTG                                       CTG CAA CGA CTG TTG CCA TGA GAC AAG GAC                                       GGT ATC CTG AAA AAC TGG AAA GAA GAA TCT                                       CCA TAG GAC TTT TTG ACC TTT CTT CTT AGA                                       GAC CGT AAA ATC ATG CAG TCT CAG ATC GTT                                       CTG GCA TTT TAG TAC GTC AGA GTC TAG CAA                                       TCT TTC TAC TTC AAG CTG TTC AAA AAC TTC                                       AGA AAG ATG AAG TTC GAC AAG TTT TTG AAG                                       AAG GAC GAC CAG TCT ATC CAG AAA TCT GTT                                       TTC CTG CTG GTC AGA TAG GTC TTT AGA CAA                                       GAA ACT ATC AAG GAA GAC ATG AAC GTT AAG                                       CTT TGA TAG TTC CTT CTG TAC TTG CAA TTC                                       TTC TTC AAC TCT AAC AAG AAA AAG CGT GAC                                       AAG AAG TTG AGA TTG TTC TTT TTC GCA CTG                                       GAC TTC GAA AAG CTT ACT AAC TAC TCT GTT                                       CTG AAG CTT TTC GAA TGA TTG ATG AGA CAA                                       ACT GAC CTT AAT GTA CAG CGT AAA GCT ATC                                       TGA CTG GAA TTA CAT GTC GCA TTT CGA TAG                                       CAT GAA CTG ATC CAG GTT ATG GCT GAA CTG                                       GTA CTT GAC TAG GTC CAA TAC CGA CTT GAC                                       TCC CCG GCT GCT AAA ACT GGT AAG CGT AAA                                       AGG GGC CGA CGA TTT TGA CCA TTC GCA TTT                                       AGA TCT CAG ATG CTG TTC CGT GGT CGT CGT                                       TCT AGA GTC TAC GAC AAG GCA CCA GCA GCA                                       GCT TCT CAG TAA                                                               CGA AGA GTC ATT                                                               __________________________________________________________________________

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a figure illustrating construction scheme of plasmid vectorpIN5T4N143 used for transformation of Escherichia coli to produce GIF143which will be purified by the method of the present invention.

FIG. 2 is a flow diagram of a preferred embodiment of the gammainterferon purification process of the present invention.

FIG. 3 is a flow diagram of a more preferred embodiment of thispurification process showing primarily chromatographic purificationmeans.

FIG. 4 is a flow diagram of a particularly preferred embodiment of thepresent invention.

FIG. 5 is a photograph which shows the protein bands of SDS-PAGEobtained by disrupting the cells (W3110/pIN5T4N146) in a buffer solutioncontaining a different concentration of zinc chloride and subjecting thesupernatant to SDS-PAGE.

FIG. 6 is a photograph which shows the protein bands of SDS-PAGEobtained by using copper sulfate in the place of zinc chloride.

FIG. 7 is a photograph which shows the protein bands of SDS-PAGEobtained by disrupting the cells (W3110/pIN5T4N143) in a buffer solutioncontaining a different concentration of zinc chloride and subjecting thesupernatant to SDS-PAGE.

The production is explained with reference to FIG. 1, in which the DNAfragment obtained by AatII and BglII digestion of pGIF54, which isessentially the same plasmid as pGIF4 disclosed in Japanese PatentPublic Disclosure No. 201995/1983, is further treated with AvaII toobtain an AvaII-BglII DNA fragment as shown in FIG. 1. Then, theintended plasmid pIN5T4N143 is obtained by inserting in the presence ofa DNA ligase a synthetic linker DNA fragment represented by

    5'--AATTCATGCAG--3'

    3'--GTACGTCCTG--5'

between an AvaII site of the AvaII-BglII fragment above and an EcoRIsite of a longer DNA fragment bearing a tetracycline resistant gene(TC^(r)) which is obtained by treating pIN5T4 (disclosed in EuropeanPatent Application 0,134,673) with Bg1II and EcoRI. The resultingplasmid contains a gene coding for a polypeptide GIF143 corresponding toGIF146 from which a sequence of 3 amino acid residues, i.e. Cys-Tyr-Cys,at the N-end of GIF146 is eliminated. Subsequently, a host (E. coliW3110) is transformed with the plasmid according to a conventionalmethod to obtain a GIF-producing transformed Escherichia coli(W3110/pIN5T4N143).

In all figures, the purification process starts with the removal ofnucleic acids from the supernatant resulting from centrifugation ofhomogenized gamma interferon-containing cells, prior stes being shownfor clarity but not being part of the present invention.

Although a purification will be described by using zinc chloride as thesalt of zinc in the Examples, the invention is not limited to this salt.Salts of zinc such as zinc sulfate and zinc acetate and salts of coppersuch as copper sulfate are also desirably used. Table I shows theprotease-inhibition effects of various metallic salt compounds, whichwere investigated by disrupting cells of the recombinant bacteria inbuffer solutions containing 1 mM or 0.2 mM of the metallic compound andmeasuring the stability of a polypeptide having h-IFN-γ activitycontained in the supernatant liquid as an indicator of the effects. Asindicated in Table I, it was found that zinc sulfate, zinc acetate, zincacetylacetonate and copper sulfate provide the desired effects as wellas zinc chloride.

                  TABLE I                                                         ______________________________________                                        Protease decomposition-inhibiting effect upon                                 polypeptide by addition of Zn, Cu and other metal salts                                      Decomposition-inhibiting                                                      effect on polypeptide                                          Metal Salt       1 mM      0.2 mM                                             ______________________________________                                        None             -         -                                                  Zinc chloride    +         -                                                  Zinc sulfate     +         -                                                  Zinc acetate     +         -                                                  Zinc acetylacetonate                                                                           +         -                                                  Zinc salicylate  ±      -                                                  Copper sulfate   ++        +                                                  Ferrous sulfate  -         -                                                  Cobalt chloride  -         -                                                  Ammonium molybdate                                                                             ±      -                                                  ______________________________________                                         + having decompositioninhibiting effect                                       - not having decompositioninhibiting effect                              

FIG. 5 (photograph) is an SDS-PAGE pattern showing the stability of thepolypeptide having h-IFN-γ activity (the band of the protein shown by anrrow marked as GIF146 in the figure). The test samples were prepared bydisrupting the cells in a desired buffer containing a differentconcentration of zinc chloride. In FIG. 5 it can be seen that thepolypeptide exhibiting h-IFN-γ activity is stable in the presence of 0.5mM--2 mM of zinc chloride and susceptible to decomposition in theabsence or a lower concentration of zinc chloride. The band of theprotein shown by arrow "B" shows the polypeptide obtained bydecomposition of GIF146 with protease during course of purification. Thesample shown by 2-1 was prepared by the treatment described above byusing 2 mM of ZnCl₂ and then further proceeding purification.Furthermore, the sample shown by 2-2 was prepared by proceeding thepurification after the treatment without using ZnCl₂. The results of asimilar experimental run in which copper sulfate was used instead ofzinc chloride are shown in FIG. 6 (photograph). In this case, thedesired results are observed in the range of 0.1 mM-4 mM. Although thesesalts show sufficient protease inhibition with higher concentrations,they are preferably used in as low a concentration as possible becauseof the necessity to remove them in the purification process. It ispreferable that zinc chloride is used in the range of 1-3 mM and coppersulfate in the range of 0.25-1 mM.

FIG. 7 (photograph) was obtained with respect to a sample prepared bytreating GIF143-producing bacteria in the same manner as for FIG. 5. Inthe figure, the band of the protein shown by an arrow indicated asGIF143 is the polypeptide having h-IFN-γ activity and "E" shows thepolypeptide obtained by partially decomposing GIF143 with protease.

The primary classes of contaminants in the disrupted cell/gammainterferon mixture are small-size particulate matter and water-solublefractions such as nucleic acids, proteases, cell proteins,carbohydrates, lipids, cleaved interferon fragments and interferonaggregates and other fragments resulting from disruption of the cell inwhich the interferon was produced. We have now discovered that gammainterferon can be obtained in high purity, with the retention ofbiological activity and with good yields, by processing theinterferon-containing mixtures in a specific sequence as described belowto minimize degradation of the interferon and to remove the contaminantsfrom the interferon-containing mixture in a defined order.

Substantially improved purity and activity are obtained by removing thecontaminants in the interferon-containing mixture in the followingorder:

(1) nucleic acids;

(2) negatively charged proteases and contaminating cell proteins;

(3) positively charged proteases and contaminating cell proteins; and

(4) cleaved and aggregated interferon.

This sequence of steps is critical to obtaining the desired results ofthis invention. Provided that the listed contaminants are removed in thespecified sequence, additional steps may be used to remove othercontaminating materials such as high molecular weight hydrophobicmaterials, if present. These other materials may conveniently be removedeither after step 3 or after step 4.

There are numerous methods, known to the art, to accomplish each ofthese separations. As stated above, those methods which can accomplishthe separations under the mildest conditions, to minimize degradation ofthe interferon, are the most desirable.

We have found that a preferred method is to use an initialpolyethyleneimine precipitation followed by several chromatographicseparations to remove the contaminants in the order sepcified above. Theresins used in the chromatographic separations and the order of theiruse is as follows:

(1) anion exchange resin;

(2) cation exchange resin; and

(3) molecular sieve.

In addition to the chromatographic separations, it is useful to employprecipitation, filtration, concentration and dialysis procedures.

In a preferred procedure the gamma interferon-containing mixture issubjected to the following procedures:

(1) nucleic acid removal using polyethyleneimine precipitation;

(2) negatively charged protease and contaminating cell protein removalusing weakly basic anion exchange resin;

(3) positively charged protease and contaminating cell protein removalusing weakly acidic cation exchange resin; and

(4) cleaved and aggregated interferon and cell fragment removal using amolecular sieve.

Filtration after each step, concentration after steps 3 and/or 4 anddialysis after step 5, are useful adjunctive procedures.

This novel procedure has consistently produced gamma interferon having apurity of at least 95% and a yield in excess of 5%.

An important feature of the present invention is the novel purificationscheme, which is suitable for use with gamma interferon produced in anyone of a number of ways such as from human cells grown in tissueculture, from leukocytes collected from blood samples or through cloningtechniques well known in the art. The purification scheme isparticularly well suited for the purification of recombinant gammainterferon recovered from E. coli cells. The cells are inactivated byone of the standard methods, such as by the addition of a chemical killagent such as chlorhexidine gluconate. The inactivated cells arecentrifuged, resuspended in a buffer and homogenized. A convenientmethod for homogenization of the gamma interferon-containing cells ishigh shear disruption using a Manton-Gaulin homogenizer. The componentsof the disrupted cells are separated by centrifugation into aprecipitate and supernatant. The supernatant from this process is asuitable source for gamma interferon to be isolated and purified by themethod described herein.

The suspension of the lysed cells comprises proteins, lipids,carbohydrates and nucleic acids and insoluble cellular debris. Usingconventional procedures, the water-insoluble components are separatedfrom the water-soluble fraction of the cell which remains in thesupernatant.

It is sometimes desirable to provide certain preliminary processingsteps prior to the extraction of the gamma interferon from the cells,such as procedures to minimize degradation of the interferon duringprocessing. Any such preliminary processing steps may be used providedthey do not interfere with the purification scheme described herein.

The multistep purification scheme achieves superior yields of pureinterferon while maintaining biological activity. The sequence ofseparation steps is highly significant and is critical to achieving thedesirable results disclosed.

The order of removal of the contaminants from the interferon-containingmixture is as follows:

(a) removal of nucleic acids;

(b) removal of negatively charged proteases contaminating cell protein;

(c) removal of positively charged proteases and contaminating cellprotein;

(d) removal of low and high molecular weight impurities, cleavedinterferon and interferon aggregates.

For reasons presently unknown, removal of impurities in the order statedis critical to achieving high yields of purified gamma interferon withretention of biological activity. The individual steps used for theremoval of each class of impurities are conventional and known to theart. Due to the tendency of the gamma interferon to cleave or aggregateinto inactive forms under harsh processing conditions, purificationssteps which can be conducted under the mildest processing conditions arepreferred.

The invention is further described utilizing specific processing stepsand conditions which have been found to minimize degradation of theinterferon, but it should be recognized that other conventionalprocessing steps may be substituted for those disclosed provided thatthe sequence of impurity removal remains as described.

Unless otherwise stated in the following description, pH values givenmay generally vary ±0.5, preferably in the range ±0.25 and mostpreferably ±0.1. Conductivity measurements may generally vary ±5 mS, andare preferably held in the range ±3 mS. Operations are performed at atemperature in the range of from about 2° to about 15° C.

The first step of the processing scheme involves the removal of nucleicacids. This removal is conveniently accomplished by addingpolyethyleneimine to the supernatant from the centrifuged mixture oflysed gamma interferon-containing cells. Alternatively, thepolyethyleneimine solution may be added prior to homogenization of thecells, if desired. The polyethyleneimine is added slowly with stirringto a maximum concentration of about 0.8% and the mixture is allowed tosettle for an appropriate period, generally in the range of from about30 to about 90 minutes. The mixture is then centrifuged and thesupernatant collected. Excellent results are obtained when thepolyethyleneimine is added as a 10% (v/v) solution in H₂ O in amountsufficient to result in the polyethyleneimine consisting of from about0.7 to about 0.8% (v/v) of the total solution. The pH of the solution is8±0.5, preferably ±0.1 and the temperature is held in the range of fromabout 2° to about 15° C. The protein concentration in the supernatant isdetermined at this stage and at each further processing stage by thestandard Coomassie blue binding assay.

Another procedure for removal of the nucleic acid is by usingchromatography on hydroxyapatite or immobilized PEI. Precipitation withprotamine sulfate is another useful procedure.

After removal of the nucleic acids, the gamma interferon-containingmixture is subjected to a first protease removal step. The mostconvenient method for removing the proteases is by chromatography of thesupernatant from the nucleic acid removal step utilizing an anionexchange resin. Quaternary aminoethyl, mixed amine or other intermediatebase resin or a weak base resin such as p-amino benzyl cellulose isparticularly useful.

Quaternary aminoethyl is a preferred anion exchange resin. Thequaternary aminoethyl may be attached to a cross-linked dextran,cellulose, agarose or acrylic support. The pH of the supernatant liquidis adjusted to 8.7±0.5, preferably ±0.1, utilizing sodium hydroxide orany other convenient base. The conductivity of the solution is adjustedto below 10 mS, preferably in the range of from about 4 to about 8 mS,by the addition of deionized H₂ O.

The elution buffer comprises 20 mM sodium4-(2-hydroxyethyl)-1-piperazine-propane sulfonate and 0.1% (v/v)2-mercaptoethanol. The pH of the buffer is adjusted to approximately 8.7with sodium hydroxide or other base. Other buffers suitable for use inthe same pH range may be substituted for the piperazine derivative andother antioxidants may be substituted for the mercaptoethanol.

The quaternary aminoethyl column is pre-equilibrated with the buffersolution, the gamma interferon-containing solution is added and theadsorbed material eluted with the same buffer. Approximately the firsttwo-thirds of the eluted protein solution, i.e., the first two-thirds ofthe volume, is pooled for transfer to the next purification step. Theremaining one-third of the eluate may be rechromatographed on the samecolumn equilibrated in the same manner. Approximately the firsttwo-thirds of the protein flow-through is again pooled. The remainingsolution may be further processed in the same manner. As previously, theprotein concentration is determined by a Coomassie blue binding assay.

An optional concentraton step may be employed at this point in thepurification. One convenient method of concentrating the solution isprecipitation with ammonium sulfate. The eluate from the quaternaryaminoethyl column is passed through a 0.2μ filter and ammonium sulfateis added to a final concentration of from about 40 to about 60%saturation, with stirring, over a 5-10 minute period. The suspension isallowed to stand for several hours in an ice bath. The precipitate isthen collected by centrifugation and may be stored at approximately -20°C. until required for further processing.

When required, the precipitate is dissolved in a solution comprising 20mM Tris-HCl and 0.1% 2-mercaptoethanol at a pH of approximately 7.5 thathas been previously passed through a 10,000 molecular weight cut-offfilter. The conductivity of the solution is lowered to from about 3 toabout 5 mS by the addition of a solution comprising 10 mM Tris-HCl and0.1% 2-mercaptoethanol at a pH of approximately 7.5. The solution ispassed through a 0.2μ filter and is ready for further processing. Otherbuffers suitable for use in the same pH range may be substituted for theTris-HCl and other antioxidants may be substituted for themercaptoethanol.

The positively charged proteases and other proteins in the solution areremoved in the next processing step, which is conveniently accomplishedutilizing a cation exchange resin.

Excellent results have been obtained using a carboxymethyl cationexchange resin (carboxymethyl) attached to cross-linked dextran,cellulose, agarose or acrylic support). The pH of the solution from theprevious process step is adjusted to about 7.5 utilizing HCl or otherappropriate acid. 2-Mercaptoethanol or other suitable antioxidant isadded to a concentration of about 0.1% (v/v). Deionized water containing0.1% (v/v) 2-mercaptoethanol is also added to reduce the conductivity tobelow 20 mS, preferably to the range of about 3-5 mS. The solution isfiltered through a 0.2 micron filter in preparation for subsequentchromatography.

The cation exchange resin column is equilibrated with a suitable buffersuch as a solution comprising 20 mM Tris-HCl and 0.1% 2-mercaptoethanolat a pH of approximately 7.5. After column equilibration by washsing thecolumn two or three times with the equilibrating buffer and addition ofthe gamma interferon-containing solution, the solution is eluted withapproximately 13 to 15 column volumes of a gradient of sodium chloridedissolved in the equilibrating buffer. The sodium chloride content isincreased from 0 to a maximum of approximately 0.5M in the buffer.

Appropriate fractions are collected and may be analyzed by gelelectrophoresis (SDS-PAGE), analytical HPLC and antiviral activity. Thepurest fractions are pooled for further processing. The fractionscontaining interferon of lower purity may be precipitated withapproximately 40 to 60% saturation ammonium sulfate, redissolved,filtered and rechromatographed on a carboxymethyl column as previouslydescribed. Fractions collected from the rechromatographed solution areanalyzed and the purest fractions pooled with the fractions obtainedfrom the first carboxy-methyl elution.

If the presence of high molecular weight hydrophobic impurity isdetected by SDS-PAGE or other appropriate procedure, the eluate issubjected to optional chromatography to remove such impurities at thisstage in the purification process. A phenyl resin has been found toprovide satisfactory results. Octyl and butyl resins may also be used.The solution from the previous processing step is filtered through a0.2μ filter and sodium chloride added (0.5-0.75M) to raise theconductivity of the solution to approximately 50 to 75 mS.

The buffer is a solution comprising 20 mM Tris-HCl, 0.1% (v/v)2-mercaptoethanol and 50 to 850 mM, preferably 500 to 700 mM, sodiumchloride or other salt to increase the conductivity to the appropriaterange.

The column is pre-equilibrated with the buffer and the sample is loadedonto the column. From about 2 to about 4 column volumes of the buffersolution are added to the column. The adsorbed material is then elutedwith at least one and preferably from about 5 to about 10 column volumesof a solution comprising 20 mM Tris-HCl, 100 mM NaCl and 0.1% (v/v)2-mercaptoethanol at a pH of approximately 7.5. Appropriately sizedfractions are collected and analyzed using SDS-PAGE, analytical HPLC andantiviral activity. The purest fractions are pooled.

It is generally desirable to concentrate the interferon-containingsolution after the phenyl-column chromatography. It is also generallydesirable to concentrate the interferon-containing solution at thisstage in those instances where the optional hydrophobic columnchromatography step has not been utilized.

The protein concentration of the solution is determined by the Coomassieblue binding assay. If the protein concentration is determined to beless than 0.2 mg/ml, the solution is preferably concentrated byultra-filtration employing a 10,000 molecular weight cut-off membrane.

Further concentration may be accomplished by adding ammonium sulfate tothe solution to a final ammonium sulfate concentration of from about 40to about 60% saturation with stirring over a 5 to 10 minute period. Thesuspension is allowed to stand in an ice bath after which theprecipitate is collected by centrifugation. The precipitate isredissolved in a solution comprising 20 mM Tris-HCl, 500 mM sodiumchloride and 0.1% 2-mercaptoethanol at a pH of about 7.5 that has beenpreviously filtered through a 10,000 molecular weight cut-off filter.The concentrated solution is passed through a 0.2μ filter in preparationfor the next purification step.

Low and high molecular weight impurities and cleaved gamma interferonand interferon aggregates are removed in a final chromatographicpurification step by passing the gamma interferon-containing solutionfrom the previous processing step through a gel filtration resin. Thehydrophilic filtration gel acts as a molecular sieve to separateappropriate sized fractions from high and low molecular weightimpurities contained in the solution. A particularly useful filtrationgel is a cross-linked dextran based gel, identified by the trademarkSEPHADEX G-100, manufactured by Pharmacia Fine Chemicals. The resin hasa fractionation molecular weight range of 4,000 to 150,000 for globularprotein and peptides and 1,000 to 100,000 for polysaccharides. Otherresins having cut-off ranges of from about 1,000 to about 200,000 forproteins may also be used.

The SEPHADEX G-100 resin column is pre-equilibrated with a buffersolution comprising 20 mM Tris-HCl, 500 mM NaCl and 0.1%2-mercaptoethanol at a pH of approximately 7.5. The adsorbed material iseluted with the buffer and appropriate fractions collected. The proteinconcentration of each fraction is determined by a Coomassie blue bindingassay. The fractions are combined on the basis of purity as judged bySDS-PAGE, analytical HPLC and antiviral activity.

Alternatively, the precipitate from the ammonium sulfate concentrationstep may be dissolved in a buffer solution of 20 mM sodium phosphate,500 mM sodium chloride and 0.1% (v/v) 2-mercaptoethanol at a pH of about7.5. The gamma interferon-containing solution is charged to a SEPHACRYLS-200 gel filtration column, preequilibrated with the same buffer(SEPHACRYL S-200 is a trademark of Pharmacia Fine Chemicals for a resinof agarose cross-linked with acrylamide). The final product is a clearto slightly hazy solution, colorless to light yellow in color. Theapparent molecular weight determined by SDS-PAGE is in the range of17,000 to 19,500.

The purified gamma interferon is dialysed against a buffer before use. Asuitable buffer comprises 20 mM sodium phosphate and 6 mM L-cysteine ata pH of about 6.8. Another suitable buffer is 15 mM sodium phosphate, 8mM sodium citrate and 6 mM L-cysteine HCl at a pH of 5.0. It ispreferable to continue to dialyse for 8 hours or more and tocontinuously flush nitrogen through the system to minimize oxidation.

If necessary, the purified gamma interferon solution can be concentratedin the manner described above.

The present invention will now be explained with reference to thereference example and the working example 1-3.

REFERENCE EXAMPLE: (CONSTRUCTION OF GIF143 EXPRESSION VECTOR)

A GIF143 expression vector was produced according to the followingprocedures.

pGIF54 (a plasmid equivalent to pGIF4 bearing a gene coding for GIF146)was obtained from WA802/pGIF54 which was a dcm Escherichia colitransformant (a strain lacking methylation of cytosine) according to aconventional method. pGIF54 (5 μg) was treated with 20 units of AatIIand 20 units of BglII to obtain a DNA fragment of about 600 base pairsbearing a part of GIF146 gene and a lac UV5 promoter. Then, 0.5 μg ofthe DNA fragment was cleaved by using 5 units of AvaII to obtain thefragment of about 400 base pairs bearing a part of GIF146 gene. On theother hand, 5 μg of pIN5T4 was digested by using 20 units of EcoRI and20 units of BglII to obtain the DNA fragment bearing atetracycline-resistant gene, lpp promoter, and a replication initiationsite. Both the DNA fragment and 0.5 μg of the chemically-synthesizedlinker shown in in FIG. 1 (synthesized by using a DNA synthesizer;Applied Biosystems 380A) were subjected to mixed-ligation to obtainpIN5GN143. W3110 was transformed by the obtained pIN5T4N143 by aconventional method, for example, the method described in JapanesePatent Public Disclosure No. 63395/1983, to obtain W3110/pIN5T4N143.

It was confirmed that the obtained transformant was a GIF143-producingstrain by the following procedures.

W3110/pIN5T4N143 was cultivated with shaking in 1.5 ml of a mediumcontaining polypeptone 3%, yeast extract 2%, glurose 2%, KH₂ PO₄ 0.5%,MgSO₄.7H₂ O 0.010% and tetracycline 20 μg/ml in a 16.5 mm test tube at30° C. (OD₆₆₀ =8), 0.5 ml of the culture mixture was transferred to anEppendorf cup of 1.5 ml and centrifuged to collect the cells (10,000rpm, for 5 minutes). The cells were suspended in 0.5 ml of PBS solution(0.8% NaCl, 0.02% KCl, 0.115% Na₂ HPO₄, 0.02% NaH₂ PO₄) containing 1mg/ml lysozyme 1 mM-EDTA and reacted at 0° C. for 30 minutes. The cellswere then disrupted by repeating freeze-thawing treatment 3 times andthe supernatant fraction was recovered by centrifugation (10,000 rpm,for 10 minutes). The supernatant fraction was investigated foranti-virus activity according to the method described in Japanese PatentPublic Disclosure No. 201995/1983. As a result, an anti-virus activityof 6×10⁴ units/ml was recognized. On the other hand, the cells obtainedby collection in the same manner as before were dissolved in 200 μl ofan SDS sample solution (10 mM phosphate buffer solution containing 7Murea, 1% SDS, 1% 2-mercaptoethanol, pH 7.2) and then heated on a boilingwater bath for 10 minutes. The resulting sample (20 μl) was isolated by13% SDS-PAGE and subjected to protein staining with Coomascie blue R250As a result, it was confirmed that GIF143 protein (about 18 kd molecularweight) in a yield corresponding to about 20% of the total protein ofEscherichia coli was produced.

EXAMPLE 1 Extraction and Purification of GIF146

W3110/pIN5T4 which was a GIF146 producing strain was cultured withaerating and shaking in a medium containing 3% polypeptone, 2% yeastextract, 2% glucose, 0.5% KH₂ PO₄, 0.01% MgSO₄ 7H₂ O and 20 μg/mltetracycline for 24 hours. The cells of the culture mixture werecompletely killed with chlorohexidine gluconate and centrifuged (8,000rpm, for 10 minutes) to obtain 800 g of the W3110/pIN5T4 wet cells. Theywere suspended in 5.1 liters of a cooled 20 mM tris-HCl buffer(abbreviated to "THB" hereinafter) having pH of 7.4 and containing 1 mMZnCl₂, disrupted by subjecting them to homogenizer M15 (manufactured byManton Gaulin Co., Ltd.), cooled with ice and then centrifuged (7,000rpm, for 20 minutes) to obtain the supernatant. To the supernatant wasadded an aqueous 15% solution of polyethyleneimine (abbreviated to "PEI"hereinafter) having a pH of 8.0 adjusted with HCl so as to achieve afinal concentration of 0.75%. The resulting solution was stirred for 10minutes and was allowed to stand at 4 C for 2 hours. The precipitatesproduced were removed by centrifugation (7,000 rpm, for 20 minutes) toobtain 4.7 liters of the supernatant. The supernatant was subjected to aQAE Sephadex A-25 (manufactured by Pharmacia Co., Ltd.) columnequilibrated with a 20 mM N-2-hydroxyethylpiperazinyl-N'-3-propanesulfonate buffer (EPPS buffer) of pH 8.6 to obtain the non-absorbedfraction. The fraction obtained was then subjected to a CM SepharoseCL6B (manufactured by Pharmacia Co., Ltd.) column equilibrated with 20mM THB of pH 7.4 containing 0.1% of 2-mercaptoethanol (abbreviated to"2-ME" hereinafter) and the active fraction absorbed on the column waseluted with a linear concentration gradient of 0 to 0.5M NaCl to collectfractions which had a high interferon activity. The interferon activitywas measured according to the method described in Japanese Patent PublicDisclosure No. 201995/1983. Ammonium sulfate was then added, to thefraction so as to achieve a 50% saturation and salting-out was thencarried out, followed by centrifugation at 7,000 rpm for 20 minutes toobtain the precipitate. The precipitate was dissolved in a 20 mM sodiumphosphate buffer (abbreviated to "20 mM PBS" hereinafter) of pH 7.4containing 0.3 NaC and 0.1% 2-ME and subjected to a Sephacryl S-200(manufactured by Pharmacia Co., Ltd.) column equilibrated with the samebuffer solution to obtain 298 mg of the polypeptide corresponding toGIF146 as a final purified product (the specific activity of interferon:1.6-1.7×10⁶ /mg). Analysis of the polypeptide by SDS-PAGE showed morethan 99% purity and the position of the band by the same SDS-PAGE was inagreement with that of GIF146 which was not decomposed. Furthermore, itwas confirmed that the polypeptide obtained had the same amino acidsequence as the amino acid sequence (I) by an amino acid analysis. Thisshows the usefulness of the purification method of the presentinvention.

When Sephadex G-100 (manufactured by Pharmacia Co., Ltd.) was employedinstead of Sephacryl S-200 used in the previously described purificationprocesses, similar results were obtained.

EXAMPLE 2 Purification and extraction of GIF143

In the same manner as described in Example 1, W3110/pIN5T4N143 (refer tothe Reference Example) was cultured, and the cells were killed withchlorohexidine gluconate, and collected. The wet cells (300 g) weresuspended in 2.1 liters of a 20 mM THB (pH 7.4) containing 3 mM ZnCl₂and disrupted by homogenization, followed by centrifugation (7,000 rpmfor 20 minutes) to obtain the supernatant. It was treated with PEI inthe same manner as in Example 1 to obtain 1,000 ml of the supernatant.The liquid was then absorbed on a CM Sepharose CL6B column equilibratedwith 20 mM THB (pH 7.4) containing 0.1% 2-ME and eluted by a linearconcentration gradient of 0.1-0.8M NaCl. The active fraction was dilutedto 10-fold of the original volume with 20 mM THB containing 0.1% of2-ME, absorbed on a CM-Toyopearl column (manufactured by Toyo Soda Co.,Ltd.) which had been equilibrated with the same buffer solution, wastedand then eluted with a linear concentration gradient of 0.1-0.8M NaCl.The fractions having interferon activity were collected, and afterammonium sulfate was added to the combined fractions so as to achieve a20% saturation, passed through a Butyl Toyopearl column (manufactured byToyo Soda Co., Ltd.). The fractions passed through the column werecollected and subjected to dialysis against distilled water to give 396mg of protein as a final product (interferon specific activity: 4.8×10⁶U/mg protein).

As a result of the amino acid analysis and SDS-PAGE as described inExample 1, it was found that the obtained polypeptide (GIF143) had apurity of more than 99% and had the same amino acid sequence as theabove-described amino acid sequence (III). Furthermore, none of the zinccompound added during the extraction step could be detected even byatomic absorption spectrophotometry.

EXAMPLE 3 I. Cell Harvest, Protein Release and PolyethyleneiminePrecipitation

Inactivated E. coli (W3110/pIN5T4) cells containing recombinant gammainterferon are collected by centrifugation. Cells are resuspended in 20mM Tri-HCl buffer (pH 7.5) containing 1 mM ZnCl₂. The cells aredisrupted in a high pressure homogenizer. The cell homogenate iscentrifuged and the supernatant is collected. An aqueous 10% (v/v)polyethyleneimine (PEI) solution adjusted to pH 8 with hydrochloric acidis added to the supernatant to bring the final PEI concentration to amaximum of 0.8%. The mixture is centrifuged and the supernatant iscollected.

II. Quaternary Aminoethyl (QAE) Column Chromatography

The pH of the PEI supernatant is adjusted to 8.7 with 4N NaOH. Deionizedwater is added to reduce the conductivity to below 10 mS. The batch isapplied onto a QAE column at a loading of not greater than 50 grams ofprotein per liter of gel. The column is equilibrated with a buffer of 20mM sodium 4-(2-hydroxyethyl)-1-piperazine-propane sulfonate and 0.1%2-mercaptoethanol at a pH of 8.7 prior to loading. Elution is perforemdwith the same buffer. The protein solution is collected andchromatographed in Stage III.

III. Carboxymethyl (CM) Column Chromatography

The pH of the protein eluate from Step 2 is adjusted to 7.5 with 4N HCland 2-mercaptoethanol is added to a final concentration of 0.1%. Theconductivity is adjusted to 20 mS or below by diluting withultrafiltered water containing 0.1% 2-mercaptoethanol. The solution ispassed through a 0.2μ filter and charged onto a CM column at a loadingof not greater than 35 grams of protein per liter of gel. The column isequilibrated with a buffer of 20 mM Tris-HCl and 0.1% 2-mercaptoethanolat a pH of 7.5 adjusted to a conductivity of 20 mS or below with sodiumchloride prior to loading. The column is washed with at least 2 columnvolumes of the equilibrating buffer. The gamma interferon is eluted witha salt gradient in the range of 0-0.5M NaCl dissolved in theequilibrating buffer. Fractions are combined as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

IV. Phenyl Column Chromatography

Chromatography on a phenyl column is performed after the presence ofhigher molecular weight impurities is detected by SDS-PAGE. Sodiumchloride is added to bring the conductivity to 50-75 mS before thesolution is charged onto a phenyl column at a loading of not greaterthan 15 grams of protein per liter of gel. The column is equlibratedwith a buffer of 20 mM Tris-HCl, 0.5M NaCl, 0.1% 2-mercaptoethanol at apH of 7.5 prior to loading. After the sample is loaded onto the column,it is followed by at least one bed volume of equlibrating buffer. Thecolumn is eluted with 20 mM Tris-HCl, 0.15M NaCl, 0.1% 2-mercaptoethanolat a pH of 7.5. Active fractions are combined as determined by SDS-PAGEand antiviral assay.

V. Ammonium Sulfate Precipitation

If the protein concentration of the combined carboxymethyl (Step III) orphenyl (Step IV) fractions is less than 0.2 mg/ml, the solution isconcentrated by ultrafiltration employing a 10,000 M.W. cut-offmembrane. Ammonium sulfate is added to a final concentration of 40 to60% saturation. The precipitate is collected by centrifugation andstored at about -20° C., if required.

VI Sephadex G-100 Column Chromatography

The ammonium sulfate precipitate is dissolved in a buffer of 20 mMTris-HCl, 0.5M NaCl, 0.1% 2-mercaptoethanol at a pH of 7.5. The solutionis centrifuged prior to passing through a 0.2μ filter. The filteredsolution is charged onto a Sephadex G-100 column pre-equilibrated withthe same buffer. The loading is not greater than 3.5 grams of proteinper liter of gel. The column is eluted with the same buffer andfractions are combined as determined by SDS-PAGE.

VII. Purified Gamma Interferon Dialysis

The combined Sephadex G-100 fractions are dialyzed against 15 mM sodiumphosphate, 8 mM sodium citrate, 6 mM L-cysteine HCl at a pH of 5.0.Dialysis is carried out with continuous sparging of nitrogen through thebuffer with two changes of buffer at a minimum of five hour intervals.If necessary, the dialyzed solution is concentrated by ultrafiltrationusing a 10,000 molecular weight cut-off membrane to a proteinconcentration greater than 1 mg/ml. The solution of purified gammainterferon is passed through a 0.2μ filter and stored at about -20° C.or below.

The purified gamma interferon may be stored for at least several monthsat temperatures of approximately -20° C. to about -30° C. by adding 50%glycerol to the gamma interferon-containing solution.

The gamma interferon is prepared for use by filtering through a 0.2μfilter and dialyzing the solution against a solution comprising 20 mMsodium phosphate and 6 mM L-cysteine at a pH of about 6.8.Alternatively, the dialysis solution is 15 mM sodium phosphate, 8 mMsodium citrate and 6 mM L-cysteine HCl, at a pH of about 5. After adialysis period of at least 8 hours carried out under continuousnitrogen sparging, the solution is preferably filtered through a 10,000molecular weight cut-off filter.

Product obtained has a purity of at least 95% gamma interferon and ayield in excess of approximately 5%.

We claim:
 1. A method of purifying gamma interferon from a culturemixture of a microorganism obtained by a recombinant DNA technique andcapable of producing gamma interferon, wherein a zinc salt or coppersalt and polyethyleneimine are added during a step of extraction orpurification of gamma interferon.
 2. A method according to claim 1,wherein the microorganism cells recovered from the culture mixture ofthe microorganism capable of producing gamma interferon are suspended ina solution containing a zinc salt or copper salt, and disrupted, andpolyethyleneimine is added to the supernatant obtained by centrifugationof the suspension of the disrupted cells.
 3. A method according to claim1 wherein said gamma interferon is human gamma interferon.
 4. A methodaccording to claim 2 wherein said zinc salt is added in such an amountthat the concentration thereof is in the range from 0.5 mM to 5 mM; orsaid copper salt is added in such an amount that the concentrationthereof is in the range from 0.05 mM to 3 mM; and polyethyleneimine isadded in such an amount that the concentration thereof is in the rangefrom 0.5 to 1.5%.
 5. A method of claim 2 which comprises the furthersteps of sequentially removing negatively charged contaminatingproteins; removing positively charged contaminating proteins; andremoving low and high molecular weight materials from the gammainterferon-containing solution by the steps of sequentially removing(1)negatively charged contaminating proteins by column chromatography witha weak base anion exchange resin; (2) positively charged contaminatingproteins by column chromatography with a weak acid cation exchangeresin; (3) low and high molecular weight materials by permeationchromatography with a gel filtration resin.
 6. The method of claim 5where the process includes the additional step of removing highmolecular weight hydrophobic materials from the gammainterferon-containing solution either immediately after removal of thepositively charged proteins or immediately after removal of the low andhigh molecular weight materials.
 7. The method of claim 5 wherein theanion exchange resin is a quaternary aminoethyl resin.
 8. The method ofclaim 5 wherein the weak acid cation exchange resin is a carboxymethylresin.
 9. The method of claim 5 wherein the filtration gel is across-linked dextran based gel or a resin of agarose cross-linked withacrylamide.
 10. The method of claim 9 wherein the anion exchange resinis a quaternary aminoethyl resin and the weak acid cation exchange resinis a carboxymethyl resin.
 11. The method of claim 5 further comprisingconcentration of the gamma interferon-containing solution after step 1,after step 2, or after each of step 1 and step 2, wherein the gammainterferon-containing solution is concentrated by ultrafiltration, byprecipitation with ammonium sulfate, or by ultrafiltration followed byprecipitation with ammonium sulfate.
 12. The method of claim 10 furthercomprising the concentration of the gamma interferon-containing solutionafter anion exchange, after cation exchange, or after each of anionexchange and cation exchange by ultrafiltration, by precipitation withammonium sulfate, or by ultrafiltration followed by precipitation withammonium sulfate.
 13. The method of claim 5 where the process includes afinal step of dialysis against cysteine-containing buffer in anoxygen-free environment of the gamma interferon-containing solution. 14.A method of claim 2 comprising the further steps of:(1) centrifuging themixture containing polyethyleneimine and zinc or copper salt to separatethe resulting precipitate from the supernatant solution; (2) adsorbingthe solution from step 1 on a column containing an anion exchange resin;(3) eluting the adsorbing material; (4) adsorbing the eluate from step 3onto a column containing a cation exchange resin; (5) eluting theadsorbed material; (6) adsorbing the eluate from step 5 onto a columncontaining a gel filtration resin; and (7) eluting the adsorbedmaterial.
 15. The method of claim 14 wherein the material adsorbed onthe anion exchange resin is eluted with a buffer comprising sodium4-(2-hydroxyethyl)-1-piperazine-propane sulfonate and 2-mercaptoethanol.16. The method of claim 14 wherein the material adsorbed on the cationexchange resin is eluted with a buffer comprising Tris-HCl and anantioxidant.
 17. The method of claim 14 wherein the material adsorbed onthe gel filtration resin is eluted with a buffer comprising Tris-HCl.18. The method of claim 15 wherein the material adsorbed on the cationexchange resin is eluted with a buffer comprising Tris-HCl and anantioxidant and wherein the material adsorbed on the gel filtrationresin is eluted with a buffer comprising Tris-HCl.
 19. The method ofclaim 18 wherein the anion exchange resin is a quaternary aminoethylresin; the cation exchange resin is a carboxymethyl resin; and the gelfiltration resin is Sephadex G-100.
 20. A method according to claim 3wherein the human immune interferon is GIF146 or GIF143.
 21. The methodof claim 12 wherein the process includes a final step of dialysis of thegamma interferon-containing solution against cysteine-containing bufferin an oxygen-free environment.